Antimicrobial resistance profiles of and associated risk factors for Pseudomonas aeruginosa nosocomial infection among patients at two tertiary healthcare facilities in Lusaka and Copperbelt Provinces, Zambia

Abstract Background Antimicrobial resistance (AMR) of pathogens such as Pseudomonas aeruginosa is among the top 10 threats to global health. However, clinical and molecular data are scarce in Zambia. We, therefore, evaluated the AMR profiles of P. aeruginosa nosocomial infections (NIs). Methods A year-long hospital-based cross-sectional study was conducted at two large tertiary-level hospitals in Zambia. Patients with current or previous hospital contact were screened for NIs. The current study focused on patients diagnosed with P. aeruginosa NIs. Clinical specimens were collected for bacteriological culture, and PCR amplification of 16S rRNA gene fragments was performed on pure isolates. Hospital or NIs were defined as infections that arise during hospitalization, occurring at least 48 h after admission. The Kirby–Bauer’s disk diffusion method was used to evaluate antibiotic resistance patterns. The association between AMR and risk factors was analysed using the χ2 test. Results Eight hundred and forty-one patients were screened, and clinical specimens were collected and analysed. Of them, 116 (13.7%) were diagnosed with P. aeruginosa NIs. The participants’ ages ranged from 15 to 98 years, with a mean of 51 (SD ± 18). Catheter-associated urinary tract infections (57%) were the most common, followed by pressure sores (38.7%). P. aeruginosa isolates were primarily susceptible to amikacin, which had the highest resistance to FEP. We observed a high prevalence of multidrug resistance (73.6%). The AMR was associated with carbapenem-hydrolysing β-lactamase gene blaOXA-51 and surgical care. Conclusions This study has demonstrated that multidrug-resistant P. aeruginosa is prevalent in hospitals in Zambia’s Lusaka and Ndola districts and possibly countrywide.


Introduction
The discovery of PEN at St Mary's Hospital in London in 1928 by Alexander Fleming was a breakthrough in medical science and practice.This discovery led to the introduction of antibiotics, significantly reducing the number of infection-related deaths.Therefore, predictions were made that infectious diseases would finally be eliminated as more antimicrobial compounds were discovered. 1 Unfortunately, developing and rising antimicrobial resistance (AMR) to these antibacterial agents quickly diminished this optimism. 2Since then, the WHO has declared AMR as one of the top 10 global public health threats facing humanity. 3It is estimated that AMR will lead to 10 million annual human deaths globally by 2050 if no interventions are implemented to combat it at global, regional and national levels. 4Despite scanty information in Africa, the available data show evidence of increasing trends in AMR, suggesting that the region shares the worldwide trend of this problem. 5ram-negative bacteria, especially non-fermenting pathogens, have been implicated in nosocomial infections (NIs).Pseudomonas aeruginosa, one of the most common hospital pathogens, is a causative agent of many severe opportunistic infections, particularly in immunocompromised patients. 6High morbidity and mortality occur due to weakened host defences, bacterial resistance to antibiotics and the production of extracellular enzymes and toxins. 7β-Lactamase production has significantly contributed to the rise in resistance to beta-lactam antibiotics, becoming a public health threat. 8The emergence and the spread of metallo-β-lactamases, especially in nonfermenters like P. aeruginosa, have become therapeutic challenges.Class C β-lactamases (Amp C) confer resistance to the same antibiotics as ESBLs with additional resistance to cephamycin and β-lactamase inhibitors. 9Infections caused by AMR bacteria pose serious challenges that include prolonged hospital stays, higher hospital costs and poorer clinical outcomes in that they cause severe morbidities and mortality. 9,10ue to the need for country-specific data on the burden of AMR and the factors driving its spread, the Zambia National Public Health Institute recently developed an integrated AMR surveillance framework to guide the fight against AMR. 11A recent study by Kaluba et al. 12 found low carbapenem resistance in P. aeruginosa.It recommended improved AMR surveillance, antimicrobial stewardship and infection prevention and control at the University Teaching Hospital (UTH) in Zambia.Nevertheless, specific clinical and molecular AMR data on resistant P. aeruginosa, such as data on associated genes, are scarce. 11,12Therefore, this study evaluated antimicrobial and molecular profiles of P. aeruginosa from patients with NIs at two sizeable tertiary healthcare facilities in Zambia.

Study site and design
A hospital-based cross-sectional study was conducted between April 2020 and April 2021 at the UTH in Lusaka district and the Ndola Teaching Hospital (NTH) in Ndola district, Zambia.The UTH is a 2000-bed capacity tertiary-level hospital and the largest referral centre in Zambia.At the same time, the NTH has a capacity of 948 beds and is the second largest hospital in the country after the UTH.The UTH is located in the Lusaka district, the capital city of Zambia, and serves as a national referral hospital.On the other hand, the NTH is a referral hospital mainly serving the northern part of the country.
These two teaching hospitals' size and pyramid-type referral systems make them ideal places to study NIs in Zambia.The two hospitals, located more than 320 km apart, were included as study sites to ascertain the diversity of AMR patterns in these two hospitals.

Sample size estimation
According to Gosling et al., [13][14][15] data on the prevalence of hospitalacquired infections in Zambian hospitals were scarce.In this study, we assumed an estimated prevalence of 10% positivity to P. aeruginosa among patients screened for NI, 16 a 95% confidence level of obtaining a true estimate and an allowable error of 2%.Based on these assumptions, we used the AusvetEpitool (https://epitools.ausvet.com.au/oneproportion) to estimate the sample size of 841 patients.Considering the size of the two hospitals, sample estimates were distributed proportionally according to the bed capacity.Therefore, we set to screen three quotas (640) and one quota (201) of patients at the UTH and NTH, respectively.

Sampling
In this study, we applied consecutive sampling.We included everyone who met the inclusion criteria as they attended the hospitals (UTH and NTH) either as returning outpatients or inpatients.All consecutive (until meeting sample size) P. aeruginosa suspected infected patients meeting the eligibility criteria were identified and enrolled after informed consent.

Patient selection
Participants were enrolled among adult medical and surgical patients after obtaining consent from patients or their next of kin.Hospital or NIs were defined as infections that arose during hospitalization, occurring at least 48 h after admission. 17The history of prior hospital contact within a month was also documented.Patients who developed clinical evidence of nosocomial wounds (surgical or pressure sore) and respiratory or urinary tract infections with a positive P. aeruginosa culture were included in the study.
Patients who presented with localized swelling, pain, purulent discharge, redness or heat in the skin, subcutaneous tissue, deep soft tissue, organ, or spaces and at least one positive culture for P. aeruginosa after 48 h post-surgical intervention were considered as nosocomial surgical site infection.Patients with fever (> 38°C), dysuria, frequency, urgency or suprapubic tenderness with no other recognized cause after 48 h of admission or recent history of catheterization were also considered.Non-catheterized patients were considered to have nosocomial urinary tract infections after obtaining a positive culture from their midstream urine.
Furthermore, patients with fever (> 38°C), chills, cough or hypotension and at least one positive culture for P. aeruginosa in sputum or respiratory secretions after 48 h of admission were considered to have a nosocomial respiratory infection and included as study participants.However, those patients who could not give either consent, clinical details or samples due to different conditions were excluded from the study.

Clinical data and laboratory specimen collection
Questionnaire data were administered to enrolled patients using the Epi collect five electronic tools (https://five.epicollect.net/). 18Additional data were obtained from attending internists and surgeons for their decision if we could not select based on the above criteria.Face-to-face interviews were used to collect information on each patient's socio-demographic variables and potential risk factors for NI.For those unable to provide information, the caregiver was interviewed.Clinical data on chronic diseases, hospitalization, previous admission, ward type and antimicrobial taking history were collected by reviewing the patient's medical record and consulting the attending physician and surgeon.Clinical specimens such as urine, respiratory secretions and wound swabs were collected as soon as NI was suspected.

Wound sample collection
Amies media sterile swabs were used to collect discharge from patients' septic burns, pressure sores or other wounds and at intravenous injection sites for those in a dialysis unit.Using Levine's technique, wound/pus specimens were collected aseptically by sterile cotton swabs dipped in normal saline. 19This technique has been reported to detect more organisms in acute and chronic wounds than other techniques. 19,20

Urine sample collection
Patients suspected of non-catheterized urinary tract infection were instructed to collect 10 mL midstream clean-catch urine samples using a wide-mouth sterile container.For catheterized patients, the catheter was clamped below the port to allow for urine to collect in the tubing.The catheter was disinfected with 70% alcohol, and 10 mL of freshly voided urine was aseptically collected through the port using a syringe.The urine sample was transferred to a sterile container.While changing Ntanda Mukomena et al.
catheters, the medical device tips were put in normal saline and taken for cultures.

Respiratory sample collection
In patients with chest symptoms, we collected the 'deep cough' sample of the early morning before eating or drinking anything to avoid bias in interpreting the results.At first, the patients needed to rinse out the mouth with clear water for 10-15 s to eliminate any contaminants in the oral cavity.After expelling saliva, the patients then breathed in deeply three times to cough at 2-min intervals until bringing up some sputum.At least 5 mL of sputum was then released in a sterile, well-closed container.ICU, the tip of the endotracheal tube was aseptically submitted for culture.
The hospital and wards of origin, date and time of collection, specimen type and tests performed were carefully noted.All specimens were placed in tightly sealed, leak-proof containers and transported in sealable, leak-proof plastic bags.
After labelling sterile containers, clinical specimens were transported to microbiology laboratories at the University of Zambia (UNZA) for UTH specimens and the Tropical Diseases Research Centre for the NTH.All samples were transported for processing within 30min to 2 h of collection.

Processing, isolation and identification
All the specimens (swabs, urine and respiratory) were inoculated onto blood agar and MacConkey (Oxoid Ltd, Basingstoke, UK) and incubated aerobically at 37°C for 24 h.Urine was inoculated using the calibrated loop that measures about 1 μL, and after 24 h of incubation, plates were examined and inspected for bacterial growth.Colonies were counted using a colony counter and checked for significant bacteriuria.Cultures from catheterized and non-catheterized patients that grew ≥10 2 and 10 5 cfu/mL, respectively, were taken as considerable bacteriuria and processed further. 21ure bacterial growths from wounds, urine and respiratory samples were examined for colony morphology.All flat, large and greenish nonlactose fermenting colonies on MacConkey were sub-cultured onto nutrient agar (Oxoid Ltd) and subjected to Gram staining and conventional biochemical tests for presumptive identification.Isolates that were Gram-negative rods, oxidase-positive, catalase-positive, Simon's citrate-positive and urease-negative were considered presumptive P. aeruginosa. 22

Molecular conformation of isolates
In addition to the phenotypic analysis, molecular analysis was performed to confirm the isolates.According to the manufacturer's instructions, DNA was extracted from purified presumptive positive cultures of P. aeruginosa using commercial DNA extraction kits (Qiagen, Hilden, Germany).PCR amplification was used to amplify 16S rRNA gene fragments from purified genomic DNA of culture isolates.The PCR master mix contained 1×PCR buffer [50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris/HCl (pH 9.0), 10 µg gelatine mL −1 ], 200 µM deoxynucleotide triphosphates, 0.3 µM each of the 16S rRNA primers, 1.5 U Taq polymerase (Gibco BRL) and ∼ 10 -100 ng DNA in a final reaction volume of 100 µL.According to the manufacturer's instructions, PCR based on Extaq protocol [Takara Biotechnology (Dalian) Co, Ltd] was used for species identification using specific primers targeting the 16S rRNA gene as previously described. 23,24PCR was also performed to identify resistant genes using specific primers for Bla OXA 23, Bla OXA 51 and Bla IMP genes. 25The following thermal cycling conditions were used: 98°C for 10 s, 35 cycles of 98°C for 30 s, 54°C for 30 s and 72°C for 1 min and final extension at 72°C for 5 min.The positive amplicons were purified using Wizard SV Gel and Clean-Up System (Promega, Madison, WI, USA) according to the manufacturer's protocol.The purified DNA was later sequenced directly using a Big Dye terminator cycle sequencing ready reaction kit v3.1 and analysed on a 3500 Genetic Analyser (Applied Biosystems, Foster City, CA, USA).

Data analysis
The collected data were validated and entered into Excel Spreadsheets before exporting to STATA version 14.0 (College Station, TX: StataCorp LP, USA) for analysis.Descriptive statistics were computed and presented using words and tables.The OR and 95% CI were calculated to measure the strength of the association.
The frequency of NI was determined at a 95% CI.The proportions (%) of the different variables were estimated as the number of positive outcomes divided by the number examined.Quantitative variables were summarized using mean and SD.Frequency distributions of the variables were produced and checked for inconsistencies and input errors.A χ 2 test was performed to screen potential factors associated with AMR in univariable analysis.Predictors of AMR were determined using step-wise logistic regression.The logistic regression model was tested for goodness of fit Pseudomonas aeruginosa nosocomial infection using the Hosmer-Lemeshow test.The level of significance was set at a P value of < 0.05.The nucleotide sequences obtained from the 16S rDNA analysis were compared for similarity with other previously published sequences using the BLAST search on NCBI/PubMed https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch.

Ethical consideration
The study protocol was ethically approved by the University of Zambia Biomedical Research Ethic Committee (UNZA REC) (ref.no.671-2019).Participants provided informed written consent to collect anonymized clinical and demographic data.For illiterate participants, a witnessed fingerprint was obtained.All obtained information was confidential and only used for research or academic purposes.Electronic devices used to collect and store the data were password protected.Furthermore, authorization for data collection was obtained from all legally recognized district and hospital representatives.After clarifying the study's objective, written informed consent was obtained from adult study participants.The results were maintained anonymously and used only for the study.Positive findings were communicated to the attending clinician for appropriate treatment.

Socio-demographic characteristics
Eight hundred and forty-one patients were screened for NIs.The majority were male (81.2%).The participants ranged from 15 to 98 years, with a mean age of 51, and most (90.4%)lived in  1).The full details of these were described in a previous publication. 26The current study focussed on AMR of the 116 (13.7%) Pseudomonas aeruginosa nosocomial infection patients diagnosed with P. aeruginosa infections.Of them, 96 were male and 20 were female.Table 1 highlights the factors associated with AMR in univariable analysis.

Participant's enrolment per study sites and wards
One hundred and sixteen patients were diagnosed with P. aeruginosa NI out of 841 screened participants from the UTH (640) and NTH (201).NI types and samples collected from patients at the UTH and NTH from different departments are shown in Table 2.

Factors associated with P. aeruginosa NI
Invasive medical device-related NI was a common presentation (78.5%) among participants.However, the presence of the medical device was not associated with AMR (P value 0.4581).A urinary catheter was frequently inserted among surgical patients being managed for benign prostate enlargement (BPH).Catheterassociated urinary tract infection was the most common NI (57%).The other common diagnosis was infected pressure sores (38.7%).AMR was associated with carbapenem-hydrolysing β-lactamase gene BlaOXA-51 (P value 0.001), literacy level (P value 0.033) and surgical ward attendance (*0.014).
Factors that were not associated with AMR include having an age of ≥ 65 years, male gender, prior admission and prolonged hospital admission.More than half (62%) of participants reported a history of previous hospital exposure within the past 30 days, which were prior medical visits or being ex-bed-siders taking care of patients.

Ntanda Mukomena et al.
A high prevalence of multidrug resistance (MDR) (73.6%) and extensive drug resistance (XDR) (37.2%) was observed in the current study at all the two tertiary hospitals included in the present study (see Table 4).
The multivariate logistic regression model results showed that P. aeruginosa MDR was related to province, admission to the surgical department, current antibiotic therapy, self-medication and blaOXA 51 genes.Details of the logistic regression are shown in Table 5 (MDR) and Table 6 (XDR).

Genomic analysis
The results of amplified genes by PCR showed that six (5.1%)P. aeruginosa clinical isolates contained blaIMP.These six clinical isolates all belonged to the UTH in Lusaka and were cultured from urinary tract infection (n = 4) and skin swabs from patients with infected decubitus ulcers (n = 2).All these six patients were from the surgical outpatient department (50%) and the medical outpatient department (50%) with a history of prior medical contact within 90 days and self-medication.These clinical isolates were MDR and harboured amp C and blaOXA51 genes.Table 7 highlights the frequency of targeted resistance genes.
The results of a PCR assay for 116 clinical isolates showed that the majority 99/116 (85.3%) of P. aeruginosa clinical isolates harboured the amp C gene.These 99 clinical isolates were recovered from the UTH (90) and NTH (9) in patients presenting with urinary tract infection (49) (49.5%), infected wounds (37) (37.4%) and respiratory tract infection (5%).Figures 2-4 show agarose gel electrophoresis of the PCR product to determine the presence of PA-SS, amp C and blaOXA51 genes in P. aeruginosa clinical isolates.
The results of amplified genes by PCR showed that only eight (6%) clinical isolates contained blaOXA23.At the NTH, blaOXA23 was found in 10% of isolates from infected wounds and 6% of isolates from the UTH from patients with infected wounds (n = 2), urinary infection (n = 3) and respiratory tract infection (n = 2).

Discussion
NIs caused by antibiotic-resistant P. aeruginosa have emerged as a primary concern in clinical care settings due to the increasing development of MDR strains. 6This study evaluated AMR in patients in two tertiary hospitals in Zambia.The majority of participants were male patients with indwelling urinary catheters.In a study conducted in Ethiopia, Taye et al. 28 reported a 4.62 higher Pseudomonas aeruginosa nosocomial infection risk of developing NIs in patients with urinary catheters than noncatheterized chronic patients.This observation is consistent with other studies done in Poland, 29 China, 30 India, 31 Morocco 32 and Egypt. 33n the current study, most participants were enrolled from the UTH compared with the NTH.At the NTH, the data were collected during the pick of the COVID-19 third wave, hence the low enrolment (23.9%) of participants at this site due to a decrease in the number of patients attending hospital for non-COVID 19-related illnesses during the study period.Similarly, Quadros et al. 34 linked the reduction of patients attending hospitals with non-COVID-19-related diseases during an outbreak to public anxiety about acquiring the viral infection in the hospital and the subsequent risk of mortality and lockdown.
The current study found self-medication to be associated with AMR.Similarly, Miliani et al. 35 reported that AMR was associated with the common usage of antibiotics.Likewise, Leopold et al. 36 and Tadesse et al. 37 reported a high resistance level to commonly used antibiotics compared with less prescribed antibiotics in sub-Saharan Africa.Despite the development by the WHO of the Access, Watch and Reserve classification system to guide the use of antibiotics and prevent AMR, 38 there are numerous challenges in the implementation in various countries due to a lack of awareness, limited resources and capacity to implement the framework in many countries, especially in lower-and middle-income countries, including Zambia.This could be due to the low availability of affordable and good-quality antibiotics, leading to the inappropriate use of antibiotics and AMR. 39The association between self-medication and AMR observed in our study could also result from repeated exposure to antimicrobial agents through over-the-counter access to these drugs. 40,41he current study found high resistance levels to CAZ and ATM despite the report that P. aeruginosa is naturally susceptible to carboxypenicillins, ceftazidime and aztreonam. 6This could be explained by the high prevalence of Amp C β-lactamase in the current study.
The present study also observed a high resistance level to piperacillin despite being combined with a β-lactamase inhibitor (tazobactam).This could also result from the increased expression of the Amp C gene observed in our clinical isolates of P. aeruginosa.Amp C cephalosporinase activity is not inhibited by β-lactamase inhibitors used in clinical practice, such as CLA, SUL and TZB.
Resistance of P. aeruginosa to CIP is a rising problem in many parts of the world.In the current study, the resistance rate to CIP was low but higher than that reported in some countries in the Middle East. 42This was similar to the findings by Yang et al. 42 who reported low AK and CIP resistance among P. aeruginosa in South China.Due to the relatively low resistance of P. aeruginosa to fluoroquinolones among our clinical isolates, the mutation of gyrA/gyrB genes within the quinolone-resistance-determining region linked to fluoroquinolones resistance of P. aeruginosa was not investigated.
The multivariate logistic regression model in the present study showed that P. aeruginosa AMR was associated with selfmedication, surgical OPD attendance or ward admission and the carbapenem-hydrolysing β-lactamases gene blaOXA-51.Generally, self-medication has been reported to be associated with emergency of AMR in bacteria, 43,44 and thus, this practice needs public sensitization.In a related study, Yamba et al. 45 observed high morbidity and mortality rates associated with AMR pathogens in the same hospital (UTH) surgical ward, indicating the need to improve infection prevention and control in the surgical wards.

Conclusion
This study has demonstrated that multidrug-resistant P. aeruginosa is highly prevalent in hospitals in Zambia's Lusaka and Ndola districts and possibly countrywide.P. aeruginosa AMR was associated with self-medication, surgical care and carbapenem-hydrolysing β-lactamase gene blaOXA-51.This calls for the establishment and implementation of antimicrobial stewardship programmes and the strengthening of the surveillance system.

Limitations
The study was conducted at two large teaching hospitals, though including data from the regional hospital could give an idea of the actual burden of P. aeruginosa AMR countrywide.This study should expand to regional hospitals to obtain the real-burden AMR profiles.

Figure 1 .
Figure 1.Schematic diagram of the sample processing and P. aeruginosa isolation.

Table 1 .
Association of clinical and molecular characteristics with AMR to P. aeruginosa among patients treated for NI at two tertiary hospitals, Lusaka and Copperbelt, Zambia Ntanda Mukomena et al.Lusaka.Only 9.6% of participants completed tertiary and 18% (150) secondary education, while 563 achieved at least primary education.Around 6% (53) of participants reported no formal education.Only 10% (85) of participants reported being in formal employment, while the majority (756) were either in the informal sector or unemployed.Most participants (71%) were married.Of 841 screened, 588 patients (69.9%) had positive bacterial cultures (Figure

Table 2 .
NI types and samples collected from the UTH and NTH

Table 3 .
P. aeruginosa antimicrobial susceptibility and resistance patterns

Table 4 .
Distribution of MDR and XDR P. aeruginosa at the UTH and NTH

Table 5 .
Logistic regression analysis of the factors associated with P. aeruginosa NI and MDR at the UTH and NTH

Table 6 .
Logistic regression analysis of the factors associated with P. aeruginosa NI and XDR at the UTH and NTH

Table 7 .
P. aeruginosa analysis of targeted resistance genes